The present invention relates to a class of viruses called rhadino viruses, or gamma-2 herpes viruses. By way of example, but not by way of limitation, one commonly studied rhadino virus is Kaposi""s Sarcoma-Associated Herpes Virus (KSHV), which is also known as Human Herpes Virus 8 (HHV8). More specifically, the invention relates to a rhadino virus protein known as LANA and to a segment of rhadino virus DNA known as the rhodino virus cis-acting element (RVCAE). The LANA protein is encoded by open reading frame (ORF) 73 and is expressed in mammalian cells that are latently infected with KSHV.
The present invention relates to the discovery that LANA is necessary and sufficient for the efficient presistence of rhadino virus DNA in mammalian cells. The invention encompases methods and assays for determining whether a compound of interest can modulate the ability of LANA to enable the efficient persistence of rhadino virus DNA in a mammalian cell, the binding of LANA to rhadino virus RVCAE DNA, the binding of LANA to mammalian chromosomes, or the tethering by LANA of rhadino virus RVCAE DNA to mammalian chromosomes, as well as such compounds themselves. Because persistent rhadino virus infection is linked to diseases such as Kaposi""s Sarcoma (KS) and Primary Effusion Lymphoma (PEL), the invention also encompases methods and assays for determining whether a compound of interest can modulate rhadino virus-associated disease states, including but not limited to KS and PEL, as well as such compounds themselves.
The invention also relates to the discovery that the expression of LANA in mammalian cells along with the presence of RVCAE on a DNA plasmid is sufficient for the efficient maintenance of the plasmid in those cells. The invention additionally encompases heterologous DNAs that include RVCAE and a desired DNA such that the heterologous DNA effeciently persists as an episome in mammalian cells in which LANA is expressed. The invention also encompases heterologous DNAs that include RVCAE, a desired DNA, and an expression cassette for LANA, such that the heterologous DNA effeciently persists as an episome in mammalian cells. Because efficient persistance of a heterologous DNA in a mammalian cell is important for gene therapies and related techniques, the invention also encompases products and methods for gene therapies and related techniques that involve heterologous DNAs having RVCAE and a desired DNA.
The present invention is more fully described below.
Kaposi""s sarcoma (KS) associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) likely plays a central role in KS pathogenesis because KSHV seropositivity precedes KS and KSHV DNA is found in almost all KS lesions, whether or not there is coexisting human immunodeficiency virus (HIV) infection (Y. Chang, et al., Science, 266, 1865 (1994); P. S. Moore, et al., J. Virol., 70, 549 (1996); S. -J. Gao, et al., N. Engl. J. Med., 335, 233 (1996); P. S. Moore, Y. Chang, N. Engl. J. Med., 332, 1181 (1995)). KSHV is also associated with lymphoproliferative disorders including primary effusion lymphomas (PELs) and multicentric Castleman""s disease (E. Cesarman, et al., Blood, 86, 2708 (1995); M. B. Rettig, et al., Science, 276, 1851 (1997); J. Soulier, et al., Blood, 86, 1276 (1995); E. Cesarman, et al., N. Engl. J. Med., 332, 1186 (1995)).
Similar to the gamma-1 herpesvirus Epstein-Barr virus (EBV), KSHV infection in tumor tissue or lymphoma derived cell lines is predominantly latent. Latently infected cells have multiple copies of circularized KSHV DNA maintained as episomes (E. Cesarman, et al., Blood, 86, 2708 (1995); M. B. Rettig, et al., Science, 276, 1851 (1997); J. Soulier, et al., Blood, 86, 1276 (1995); E. Cesarman, Y. Chang, P. S. Moore, J. W. Said, D. M. Knowles, N. Engl. J. Med., 332, 1186 (1995); L. L. Decker, et al., J. Exp. Med., 184, 283 (1996). The EBV EBNA1 protein mediates efficient episome persistence through a cis-acting 1.8 kb EBV DNA sequence termed origin of plasmid replication (oriP) (J. Yates, N. Warren, D. Reisman, B. Sugden, Proc. Natl. Acad. Sci. USA, 81, 3806 (1984); J. L. Yates, N. Warren, B. Sugden, Nature, 313, 812 (1985); D. R. Rawlins, G. Milman, S. D. Hayward, G. S. Hayward, Cell, 42, 859 (1985)). The primate transforming herpes virus saimiri (HVS), which is a rhadino virus, also has a cis-acting sequence that enables efficient persistence of episomes in HVS infected cells (S.-H. Kung, P. G. Medveczky, J. Virol., 70, 1738 (1996)). However, KSHV has no obvious homology to the HVS cis-acting DNA and a trans-acting EBNA1 homolog or analog has not been identified in HVS or other gamma-2 herpesviruses.
KSHV open reading frame (ORF) 73 encodes the latency-associated nuclear antigen (LANA, LNA, or LNA1) which is predicted to be 1162 amino acids and lacks a known function (Y. Chang, et al., Science, 266, 1865 (1994); P. S. Moore, et al., J. Virol., 70, 549 (1996); S.-J. Gao, et al., N. Engl. J. Med., 335, 233 (1996); P. S. Moore, Y. Chang, N. Engl. J. Med., 332, 1181 (1995); J. J. Russo, et al., Proc. Natl. Acad. Sci. USA, 93, 14862 (1996); P. Kellam, et al., J. of Human Virol., 1, 19 (1997); L. Rainbow, et al., J. Virol., 71, 5915 (1997); D. H. Kedes, M. Lagunoff, R. Renne, D. Ganem, J. Clin. Invest., 100, 2606 (1997); F. Neipel, J. C. Albrecht, B. Fleckenstein, J. Virol., 71, 4187 (1997)). A homologous open reading frame exists in other gamma-2 herpesviruses such as, but not limited to, HV saimiri and MHV68. (J. C. Albrecht, et al., J. Virol., 66, 5047 (1992); H. W. Virgin IV, et al., J. Virol., 71, 5894 (1997)). LANA is reactive with most KSHV-immune sera which detect LANA in KSHV infected PEL cells and KS spindle cells (P. Kellam, et al., J. of Human Virol., 1, 19 (1997); L. Rainbow, et al., J. Virol., 71, 5915 (1997); D. H. Kedes, M. Lagunoff, R. Renne, D. Ganem, J. Clin. Invest., 100, 2606 (1997); F. Neipel, J. C. Albrecht, B. Fleckenstein, J. Virol., 71, 4187 (1997); S. J. Gao, et al., Nature Medicine, 2, 925 (1996); D. H. Kedes, et al., Nature Medicine, 2, 918 (1996); D. Jones, et al., N. Engl. J. of Med., 339, 444 (1998); R. Renne, et al., Nature Medicine, 2, 342 (1996); L. Szekely, et al., J. of General Virology, 79, 1445 (1998)).
The present invention encompases methods and assays for determining whether a compound of interest can modulate the ability of LANA to enable the efficient persistence of rhadino virus in a mammalian cell, the binding of LANA to rhadino virus RVCAE DNA, the binding of LANA to mammalian chromosomes, the tethering by LANA of rhadino virus RVCAE DNA to mammalian chromosomes, or any rhadino virus-associated disease states, including but not limited to KS and PEL, as as well as such compounds themselves. The present invention also encompases products and methods for gene therapies and related techniques involving the use of heterologous DNAs that include the rhadino virus cis-acting element (RVCAE) and a desired DNA such that the heterologous DNA effeciently persists as an episome in mammalian cells in which LANA is expressed, including heterologous DNAs that carry an expression cassette for LANA.
The invention is based, in part, on the discovery that LANA is necessary and sufficient for the efficient presistence of rhadino virus DNA in mammalian cells. The examples described infra provide the first identification of a trans-acting factor that supports episome persistence of gamma-2 herpesvirus DNA. The factor is identified as RVCAE. According to one embodiment of the invention the RVCAE element includes at least one copy of the rhodino virus terminal repeat (TR). According to another embodiment of the invention, the RVCAE element includes at least three copies of the rhodino virus terminal repeat (TR) and 0.6 kb of the rhodino virus adjacent unique DNA.
It is shown that LANA associates with KSHV episomes in interphase and on chromosomes in mitotic cells. Cell lines stably expressing LANA are shown to support episomal persistence of KSHV DNA. LANA is shown to concentrate to dots at sites of KSHV DNA or at sites of artificial episomes that contain the rhodino virus cis-acting element (RVCAE). These findings support a model in which LANA links, or tethers, KSHV DNA to chromosomes during mitosis to ensure efficient segregation of KSHV episomes to progeny cells.
The invention is described in more detail in the following sections and examples for purposes of clarity and not by way of limitation.
As used herein, the following terms and abbreviations shall have the meanings indicated below:
As used herein, the word xe2x80x9cmodulatexe2x80x9d (and its derivatives, such as xe2x80x9cmodulationxe2x80x9d) shall have its usual meaning, but shall also encompass the meanings of the words xe2x80x9cenhance,xe2x80x9d xe2x80x9cmimic,xe2x80x9d xe2x80x9cinhibit,xe2x80x9d or xe2x80x9cpreventxe2x80x9d (and their deriviatives).
As used herein, the word xe2x80x9corxe2x80x9d is intended to operate in the strictly logical sense. That is, a statement xe2x80x9cA or B is truexe2x80x9d is logically true if A is true, or B is true, or both A and B are true. Therefore, as used herein, the term xe2x80x9corxe2x80x9d shall have the same meaning as the idiomatic expression xe2x80x9cand/or.xe2x80x9d
As used herein, the term xe2x80x9cheterologous DNAxe2x80x9d is intended to refer to any DNA that is introduced into a cell.